Title
Remote Perturbations in Tertiary Contacts Trigger Lysine Ligation to the Heme Iron in Cytochrome c
Author
Jie Gu, Dong-Woo Shin, Ekaterina V. Pletneva
Year
2017
Journal
Biochemistry
Abstract
Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), Met80 ligation to the heme iron is critical for the protein’s electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, NMR, and EPR spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation and the pH-dependent changes in ligation provide detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling the native Met80 ligation to the heme iron.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Tertiary structure, Thermal stability, Chemical stability, Protein denaturation, Thermodynamics, Biochemistry